[Expression of VP1 gene and ELISA detection of antibodies against duck hepatitis virus].
OBJECTIVE
We developed an indirect enzyme-linked immunosorbent assay (ELISA) by the recombinant VP1 protein of duck hepatitis virus (DHV) expressed in Escherichia coli to detect antibodies against DHV.


METHODS
The VP1 gene of DHV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pMD18-T and pET-32a vectors to get a prokaryotic expression vector pET-32a-VP1. DHV VP1 gene was expressed and analyzed. A method of enzyme-linked immunosorbent assay (ELISA) was developed and studied.


RESULTS
We obtained the recombinant plasmid pET-32a-VP1 with correct sequence and orientation. DHV VP1 gene was expressed in E. coli BL21 (DE3) at a high level and had good immunoreactivity by SDS-PAGE and western blot. The optimum working concentration of antigen was 5.0 microg in 100 microL per well, the working concentration of serum samples was 1 : 100 dilution and the critical value was OD450 > or = 0.302 for the ELISA assay. The rate of coincidence of ELISA and virus neutralization test (VN) was 97.5% by detecting 80 serum samples. The method was specific, sensitive and could be applied for antibody detection of maternal antibody and the rule of antibody growth and decline in immunizing ducklings.


CONCLUSIONS
The ELISA method developed by the purified recombinant protein could be used to detect antibodies against DHV.
Ma
Xiuli
X
South China Agricultural University, Guangzhou 510642, China. maxiuli1978@163.com
Song
Minxun
M
Yu
Kexiang
K
Liao
Ming
M
Xin
Chao'an
C
D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology*; D003001:Cloning, Molecular; D004372:Ducks / Q000254:growth & development / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D004371:Hepatitis Virus, Duck / Q000235:genetics* / Q000276:immunology*; D007114:Immunization; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D000906:Antibodies; D014764:Viral Proteins